Analysis of methylation status of CGB genes in common non trophoblastic cancer

hCG is a complex heterodimeric glycoprotein hormone essential during pregnancy. The hormone and its unique β-subunit are recognised tumour markers involved in enabling anti-apoptotic pathways, promotion of invasion and angiogenesis, and supporting tumour proliferation leading to more aggressive cancer and poor prognosis. The limiting factor for the hCG production is the presence of the β-subunit which is encoded by 4 non-allelic genes, CGB3-9. These genes are regulated via transcription factors AP2α, SP1, OCT3/4 and possibly DNA methylation. DNA methylation is a repressive mark found in cytosines in DNA. The aim of this study is to investigate the role of DNA methylation in relation to CGB3-9 genes activation and hCG/hCGβ production in non-trophoblastic cancer cell lines. Bioinformatics methods were used to establish the region of interest and predict CpG islands (CGIs) within the promoter region of the target genes. Methylation-sensitive PCR and next generation sequencing were used to assess CGB3-9 promoter methylation in the cancer cell lines. The transcription level of CGB3-9 genes was investigated using qRT-PCR with TaqMan probes. Secretion in media of hCG/hCGβ was investigated with ELISA. The results demonstrated the presence of a CGI associated with CGB7 which matches previous reports of the transcription of the gene in normal and cancer tissue. Notably, this study confirmed previously reported hypomethylation in trophoblastic cell lines. Hypomethylation of the genes was found in HeLa cell line which matches literature reported transcription studies. However, other results were discordant or inconclusive leading to no significant correlation between methylation and transcription levels. This first sequencing study of CGB3-9 methylation in non-trophoblastic cancer cell lines was inconclusive for the role of the epigenetic mark in CGB3-9 genes. Further investigations need to be performed to elucidate the role of methylation